AN ABSTRACT OF THE THESIS OF
Michelle M. Moore
in
for the degree of
Fisheries Science
Title:
presented on
Master of Science
February 5. 1991
Host Responses of English Sole (Parophrys
vetulus) to Infection by the Monogenetic Trematode
Gyrodactylus stellatus
Redacted for Privacy
Abstract approve
»
Dr. Robert E. Olson
The infection intensity of the monogenetic trematode
Gyrodactyus stellatus on laboratory held English sole
(Parophrys vetulus) appeared to be influenced by handling
stress, substrate, and starvation.
~.
In bioassays testing
stellatus survival times in mucus and serum collected
from laboratory held sole at different times during
trematode infection, trematode survival times were significantly reduced in serum and mucus samples collected
from sole at the later, recovering stages of infection.
Ouchterlony gel diffusion tests of rabbit antiserum
against English sole serum diffused with English sole
mucus samples showed that the mucus of Q. stellatus
infected English sole contained factors antigenically
similiar to factors in English sole serum.
These factors
were not present in the mucus of uninfected sole.
Pre-
cipitin bands in the gel diffusions tests appeared to be
the strongest in mucus samples from sole at later, recovering stages of infection.
The results of the serum and mucus bioassays and the
Ouchterlony tests suggest the possible presence of resistance factors in both the serum and the mucus of English
sole at later stages of trematode infection, and that
these factors may result in recovery from the infection.
The results also suggest that resistance factors in the
mucus may originate from the serum.
This study did not
attempt to characterize these resistance factors.
Host Responses of English sole (Parophrys vetulus)
to Infection by the Monogenetic Trematode
Gyrodactylus stellatus
by
Michelle M. Moore
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirement for the
degree of
Master of Science
Completed February 5, 1991
Commencement June 1991
APPROVED:
Redacted for Privacy
Associate Professor of Fisheries in Charge of Major
Redacted for Privacy
Head of Department of Fisheries and wildlife
Redacted for Privacy
Date Thesis is Presented____~F~e~b~r~u~a~r~y~5~r_=1~9~9~1~_______
Typed by____________~M==i=c=h=e=l=l=e~M=.~M~o~o~r~e=_______________
ACKNOWLEDGEMENTS
I wish to thank the following people:
Dr. Robert
Olson, my major professor, for his support and guidance.
He was always available when I needed him;
Dr. steven
Kaattari, my minor professor, for his help with the immunological aspects of this thesis, and for turning me on
to Fish Immunology in the first place;
Drs. Larry curtis
and Mark Hixon for their input on the text;
Dr. Jim
Bertolini for help with the immunological methods;
and
Dr. Ted Dewitt for invaluable help with the statistical
analyses of my data.
Thanks also to the friends and Marine Science center
Aquarium volunteers who assisted me with the field collections under sometimes less than ideal conditions.
Special thanks to Michael Burger for his love,
support, and just the right combination of patience and
prodding.
TABLE OF CONTENTS
I.
II.
INTRODUCTION
1
MATERIALS AND METHODS
9
Collection and Maintanence of English Sole
Infection Levels
Condition Factors
Natural Gyrodactylus Stellatus Infection
Levels
Experimental Design of Handling Stress,
Substrate and Starvation Experiments
Handling Stress Experiments
Substrate Experiment
starvation Experiment
Statistical Analyses of Handling Stress,
Substrate and starvation Experiments
Laboratory Held, Uninfected English Sole
Serum and Mucus Sample Collection for
Bioassays and Immunological Tests
Gyrodactylus stellatus Immunized English
Sole
Mucus Bioassays
Serum Bioassays
Statistical Analyses of Mucus and Serum
Bioassays
Preparation of Trematode Antigen for
Agglutination and Gel Diffusion Tests
Rabbit Antiserum
Gel Diffusion Tests
Microtiter Agglutination Test
Macroscopic Slide Agglutination Tests
III.
RESULTS
Gyrodactylus Stellatus on English Sole
from Yaquina Bay
Handling Stress Experiments
Substrate Experiment
Starvation Experiment
Mucus Bioassays
Serum Bioassays
Gel Diffusion, Microtiter and Slide
Agglutination Tests
IV.
9
9
11
11
11
12
12
12
13
14
14
15
16
17
17
18
19
19
20
21
22
22
22
31
35
39
47
55
DISCUSSION
62
BIBLIOGRAPHY
73
LIST OF FIGURES
Figure
1
2
3
4
5
6
7
8
9
10
Regression of the mean number of worms/fish
versus the number of fish per test chamber in
the unstressed treatment of the first handling stress experiment.
24
Handling stress experiment 1; the effect of
handling stress on intensity of Gyrodactylus
stellatus on laboratory held English sole
after four weeks.
29
Handling stress experiment 2; the effect of
handling stress on intensity of Gyrodactylus
stellatus on laboratory held English sole
after four weeks.
29
Handling stress experiment 3; the effect of
handling stress on intensity of Gyrodactylus
stellatus on laboratory held English sole
after four weeks.
29
The effect of substrate on infection intensity levels of Gyrodactylus stellatus on laboratory held English sole after two weeks.
33
The effect of Gyrodactylus stellatus on survival time of unfed, newly captured, naturally infected and disinfected English sole held
in test chambers until death.
37
Mucus bioassay 1; the effect of English sole
mucus on the mean survival time of
Gyrodactylus stellatus.
41
Mucus bioassay 2; the effect of English sole
mucus on the mean survival time of
Gyrodactylus stellatus.
45
Serum bioassay 1; the effect of English sole
serum on the mean survival time of
Gyrodactylus stellatus.
49
Serum bioassay 2; the effect of English sole
serum on the mean survival time of
Gyrodactylus stellatus.
53
Figure
11
Plate 1; Ouchterlony test to detect English
sole serum factors in English sole mucus.
58
12
Plate 2; Ouchterlony test to detect English
sole serum factors in English sole mucus.
58
13
Plate 3; Ouchterlony test to detect English
sole serum factors in English sole mucus.
60
LIST OF TABLES
Table
1
2
3
4
5
6
7
8
9
Handling stress experiment 1; the effect
of handling stress on intensity of
Gyrodactylus stellatus, condition factors
and survival of laboratory held English
sole after four weeks.
26
Handling stress experiment 2; the effect
of handling stress on intensity of
Gyrodactylus stellatus, condition factors
and survival of laboratory held English
sole after four weeks.
27
Handling stress experiment 3; the effect
of handling stress on intensity of
Gyrodactylus stellatus, condition factors
and survival of laboratory held English
sole after four weeks.
28
The effect of substrate on intensity of
Gyrodactylus stellatus, condition factors
and survival of laboratory held English
sole after two weeks.
32
The effect of Gyrodactylus stellatus on survival time and condition factors of unfed,
newly captured, naturally infected or disinfected English sole held in test chambers
until death.
36
Mucus bioassay 1; the effect of English sole
mucus on the mean survival time (MST) of
Gyrodacty1us stellatus.
40
Mucus bioassay 2; the effect of English sole
mucus on the mean survival time (MST) of
Gyrodactylus stellatus.
44
Serum bioassay 1; the effect of English sole
serum on the mean survival time (MST) of
Gyrodactylus stellatus.
48
Serum bioassay 2; the effect of English sole
serum on the mean survival time (MST) of
Gyrodactylus stellatus.
52
Table
10
Results of Ouchterlony tests of English sale
mucus samples collected from two groups of
sale diffused with rabbit antiserum against
English sale serum.
57
HOST RESPONSES OF ENGLISH SOLE (PAROPHRYS VETULUS)
TO INFECTION BY THE MONGENETIC TREMATODE
GYRODACTYLUS STELLATUS
I.
INTRODUCTION
Monogenetic trematodes are small to medium sized
(.03-20 rom) ectoparasites of poikilothermous vertebrates,
usually fishes.
They have a direct life cycle on the
gills or skin of their hosts, are often highly host
specific and most are blood or mucus feeders.
The major
attachment structure is the opisthaptor, a posterior disk
which bears hooks, suckers or both (Smyth 1966, Sproston
1946, Bychowsky 1961, Dawes 1968).
The monogenetic trematode genus Gyrodactylus
in-
cludes approximately 350 species parasitic on the gills
and body surfaces of marine and freshwater teleosts (Cone
and Odense 1984).
Gyrodactylids are small (less than 1
rom), elongated monogeneans which feed on the host's epidermis and epidermal secretions (Smyth 1966).
They have
a well developed opisthaptor bearing a pair of large
anchor hooks and 15-16 marginal hooklets (sproston 1946,
Bychowsky 1961).
The subject of this study is Gyrodactylus stellatus,
a parasite of pleuronectid flatfishes (Kamiso and Olson
1986).
Like all members of the family Gyrodactylidae,
~.
2
stellatus is viviparous.
This contrasts with all other
monogenetic trematode groups which are oviparous and
possess a ciliated larval stage (Sproston 1946).
Reproduction in the genus Gyrodactylus has been described by Bychowsky (1961).
result from a single egg.
Up to four embryos may
The first born individual has
in its uterus three embryos lying one inside the other,
the second born has two embryos, the third has one, and
the fourth none.
The first three individuals differ from
the fourth in that they give birth to embryos from the
same egg cell as themselves.
In the fourth individual, a
new egg enters the uterus so that it gives birth to an
embryo arising from an egg it has produced.
That embryo
(containing three embryos) develops within 4 to 5 days
and is born, thus repeating the cycle.
The lifespan of
an individual Gyrodactylus sp. is at least 12-15 days
(Bychowsky 1961).
Lester and Adams (1974) recorded two
births per worm at 15°C with the first birth at 1.6 days
and the second birth after 6.9 days.
They recorded a
longest life-span of 28 days at 15°C and 71 days at 7°C.
Gyrodactylids do not live longer than 48 hours away from
the host and usually perish within 24 hours (Bychowsky
1961) .
Since members of the genus Gyrodactylus are viviparous and capable of rapid reproduction, they may increase to dangerous levels of infection under hatchery
3
conditions that often crowd the fish under culture
(Mizelle and Kritsky, 1967).
The only known cases of
mortality due to Gyrodactylus sp. in natural waters occurred in Atlantic salmon (Salmo salar) in Norwegian
rivers (Heggberget and Johnsen, 1982),
where~.
salaris
was thought to be recently introduced (Bakke et al.,
1990).
In this instance, high frequencies and inten-
sities of Gyrodactylus infection were seen in conjunction
with evidence of high mortalities (Heggberget and
Johnsen, 1982).
Epizootics of gyrodactylids in North American fish
hatcheries were reported as early as 1899 (Mizelle and
Kritsky, 1967).
The resulting disease condition, gyro-
dactyliasis, has caused losses in a wide variety of
captive fish species (Cone and Odense, 1984).
Losses due
to gyrodactyliasis have been reported in rainbow trout,
Onchorhynchus mykiss (formerly Salmo gairdnerii) (Mizelle
and Kritsky, 1967); black bullhead, Ictalurus melas
(Mizelle and Kritsky, 1967); golden shiner, Notemigonus
crysoleucas (Lewis and Lewis, 1970); English sole,
Parophrys vetulus (Kamiso and Olson, 1986); threespine
stickleback, Gasterosteus aculeatus (Lester and Adams,
1974); Atlantic salmon, Salmo salar (Bakke et al., 1990);
guppy, Poecilia reticulata (Scott 1985); bluegill,
Lepomis macrochiri (Hoffman and Putz, 1964); and plaice,
4
Pleuronectes platessa (Mackenzie 1970).
Gyrodactylus sp. may live almost anywhere on the
host but are usually most abundant on the fins.
The
affected surfaces may become covered with a bluish grey
slime due to increased mucus production.
When the para-
sites are very abundant, the fins can become frayed and
may eventually be eroded.
Infected fish often rub them-
selves against the sides of a tank or bottom of a pond in
an apparent effort to dislodge the parasites (Davis
1965).
English sole, Parophrys vetulus, are a major contributor to Pacific Ocean trawl fisheries off the united
states and Canada and, in Oregon, they rank third in
annual landings (Toole et al., 1987).
Juvenile English
sole utilize Yaquina Bay as a nursery ground for their
first year of life (Olson and Pratt, 1973), during which
time they are commonly parasitized by Gyrodactylus
stellatus (Olson 1978; Kamiso and Olson, 1986).
Juvenile English sole held in the laboratory have
been observed to become much more heavily infected than
those in the estuary.
Kamiso and Olson (1986) found that
fish from the estuary never averaged greater than 5.5
trematodes per fish while those held in the laboratory
were observed to undergo a logarithmic increase during
the first 9 weeks, peaking at over 1000 trematodes per
fish, then decreasing rapidly over the following 3 week
5
period.
This suggests that there may be a host response
that regulates the levels of Q. stellatus on English
sole, a response that is temporarily lost when fish are
subjected to capture and laboratory holding stresses.
In estuaries, juvenile English sole are found in
areas with sand or mud substrate (Toole et al., 1987).
Previous studies monitored Q. stellatus levels on laboratory held English sole kept in tanks without substrate
(Kamiso and Olson, 1986).
The presence or absence of
substrate may have an effect on infection intensities in
laboratory held fish.
Kamiso and Olson (1986) observed that fish dying
with heavy infections had ceased feeding and were emaciated.
They suggested that death may have been the result
of the combined effects of starvation and heavy parasitism.
They also found that the rate of parasite increase
on unfed fish was significantly higher than on fed fish,
but they did not separate the effects of starvation and
trematode infection on mortality.
The host response that regulates Q. stellatus may be
affected by the stresses associated with capture and
laboratory holding conditions.
stress is known to have
physiological effects on fish; these effects are termed
primary, secondary and tertiary.
The primary effects of
stress are increased production of corticosteroids and
catecholamines of the neuro-endocrine system (Schreck et
6
al., 1976; Schreck 1981; Mazeud et al., 1977).
These
primary responses bring about the biochemical, physiological, and immunological changes which are described as
secondary responses (Mazeaud et al., 1977).
Tertiary
responses include changes in behavior, decreased growth
rate, and increased susceptibility to disease (Wedemeyer
and McLeay 1981).
Factors known to induce a stress response in fish
include osmotic and ionic changes, pollutants, temperature changes, anesthetics and handling (Eddy 1981).
In
fish, the primary response to stress occurs quickly and
is of short duration relative to secondary and tertiary
responses (Schreck 1981; Mazeaud and Mazeaud, 1981).
In-
creased catecholamine levels occur within minutes at the
onset of stress and may last hours after cessation
(Mazeaud and Mazeaud, 1981).
The primary responses to
multiple acute handling stresses have been shown to be
cumulative and consist of stepwise increases of plasma
cortisol and glucose concentrations (Barton et al., 1986;
Flos et al., 1988).
The increased hormone levels associated with the
primary response to stress have been shown to effect
antibody producing cells.
cortisol implants resulted in
increased plasma cortisol levels, decreased levels of
antibody-secreting cells and decreased disease resistance
in juvenile coho salmon, Q. kisutch (Maule et al., 1987).
7
stress, even a relatively mild handling stress of 30
second duration, suppressed the ability of lymphocytes to
generate antibody-producing cells for at least 7 days in
spring chinook salmon, Q. tshawytscha (Kaattari and
Schreck, 1987).
Fish respond to parasitic infections by the production of antigen specific IgM-like antibodies as well
as by the elaboration of nonspecific cellular immunity,
expressed either as phagocytes (Ellis et al., 1974) or
cytotoxic cells (Evans et al., 1984); and nonspecific
soluble factors such as lysozyme (Fletcher and White,
1976), C-reactive protein (Fletcher et al., 1977), transferrin (Suzumoto et al., 1977), and interferon-like molecules (Evans and Gratzek, 1989).
In immunity to helminth
infections, the host response appears to be mediated via
antibodies, plus complement in most cases (Evans and
Gratzek, 1989).
Cellular immunity has not yet been shown
to mediate anti-helminth responses in fish (Evans and
Gratzek, 1989).
The external body surface of fish is covered by a
mucoid layer that is secreted from goblet cells in the
epidermis.
Mucus provides a mechanical and chemical
barrier to infection (Ingram 1980), and immunoglobulins
have been isolated from the mucus of some species of fish
(Bradshaw et al., 1971; Fletcher and Grant, 1969).
Nigrelli (1935) studied the effects of marine fish mucus
8
on the monogenetic trematode Epibdella melleni and found
that mucus from fish with natural immunity to the parasite had an effect on trematode survival.
Hanson (1973)
found that serum and mucus from fish species with natural
resistance decreased survival times of adult Diclidophora
embiotoci (Monogenea).
The purpose of this study was to explore the mechanism of English sole resistance to
~.
stellatus by examin-
ing the basis for the transitory loss of resistance in
the laboratory.
Specific objectives were to: Determine
if the intensity of
~.
stellatus on laboratory held fish
was influenced by handling stress, the presence or absence of substrate, and/or starvation; Determine if the
mucus layer of the fish played a role in resistance to
the trematode; and Determine if serum factors were involved in resistance.
9
II.
MATERIALS AND KBTBODS
Collection and Maintenance of English Sole:
Juvenile English sole (Parophrys vetulus) were collected from Yaquina Bay, Oregon by towing a 16 foot otter
trawl from the O.S.U. research vessel Sacajawea, or by
beach seine.
Fish were immediately transported to the
Fish Disease Laboratory at the Oregon State University
Hatfield Marine Science Center in Newport where they were
held in fiberglass tanks provided with sand filtered,
ultraviolet-light-treated salt water originating from Yaquina Bay.
Water temperature was ambient and measured
every other day.
Most sole were initially fed frozen
krill, then gradually switched to a diet of commercial
moist salmon feed over a period of three weeks.
Fish
acclimating to laboratory conditions and commercially
prepared food were fed ad libidum every other day and
were available for use in experiments.
Sole used in some
experiments were not fed after capture or were fed only
krill.
Infection Levels:
The number of trematodes per fish was determined at
the termination of each experiment, with the exception of
the starvation experiment in which sole were not fed and
10
infection levels were not determined.
Fish were trans-
ferred to individual pint-size, plastic freezer containers where they were treated for 30 minutes in a
1:5500 dilution of formalin in sea water to kill and
detach trematodes (Parker and Haley, 1960).
Following
the formalin treatment, the fish were euthanized with 2phenoxyethanol, weighed and measured.
Formalin con-
centrations were brought up to 10% in each container and
the fish in the container was examined under a dissecting
scope to make sure all trematodes were removed.
Any
attached trematodes were dislodged by a stream of formalin solution through a pasteur pipet and collected in
the container. After removal of attached trematodes, the
fish was discarded and the trematodes retained in 10%
formalin in the containers.
The number of trematodes per fish was determined by
counting the number of worms in each container.
Trema-
todes were counted in a 10-30 ml aliquot in a petri dish
backed with a transparent grid (1/4") under a dissecting
scope.
Worm numbers for each aliquot were added to get
the total number of worms per fish.
When worm numbers were too high to count directly,
estimates of total numbers were based on the number of
trematodes in three aliquots (5-30 ml) of a homogeneous
mixture of the trematodes in each container.
11
Condition Factors:
A unit-less condition factor (Fox 1962) was determined for each fish from length and weight measurements
using the equation:
CF
=
(w x 100,000)/13
where l=length and w=weight.
The condition factor was
determined for all English sole used in laboratory experiments.
Natural Gyrodactylus Stellatus Infection Levels:
Natural trematode infection levels and English sole
condition factors were determined for a collection of 60
fish captured in October 1989 and used as a basis of comparison with data from experiments.
Experimental Design of Handling stress. Substrate. and
starvation Experiments:
Experiments to assess the effect of handling stress,
substrate, and starvation on Q. stellatus infection
levels on laboratory held English sole employed an experimental design that consisted of two treatments, with
four test chambers per treatment and 10 fish per replicate.
In the third handling stress experiment, due to
the number of fish available, each treatment had three
test chambers holding 3-5 fish each.
Test chambers were
aerated, flow-through, plastic 16 x 11 x 6" containers
12
that held 5.8L each.
Fish were impartially distributed
into test chambers and the treatment each chamber was to
receive was determined with a random numbers table.
Handling stress Experiments:
The effect of handling stress was tested in several
experiments by holding test fish out water in a dipnet
for various time intervals and then allowing infections
to develop for four weeks before the number of worms per
fish and condition factors were determined.
Control fish
did not receive the stress treatment.
Substrate Experiment:
The influence of substrate on
levels on laboratory held
~.
stellatus infection
£. vetulus was tested by hold-
ing newly captured naturally infected fish in test chambers with or without a sand substrate for two weeks
before determination of the number of worms per fish and
condition factors.
The fish were fed frozen krill during
the experiment.
starvation Experiment:
To determine the effect of
~.
stellatus on the
survival of unfed English sole, survival times of naturally infected English sole were compared to those of
formalin-disinfected English sole.
The fish were not fed
13
during the experiment, and dead fish were removed and
recorded daily.
Each fish was examined under a dissect-
ing scope to confirm whether or not it was a
infected fish.
~.
stellatus
The experiment ran until no fish remain-
ed.
statistical Analyses of Handling stress. Substrate. and
starvation Experiments:
The number of worms per fish data had a Poisson distribution and were normalized with a square-root transformation.
The assumption of normality was confirmed for
the transformed trematode counts and condition factor
data with a Kolmogorov-Smirnov one-sample goodness-of-fit
test.
Student's t-test was used to determine if there were
significant differences between test treatments.
To
determine if the data from each fish in a test chamber
could be analyzed independently, a simple linear regression analysis was done to determine if the number of
worms per fish was influenced by the number of fish per
test chamber.
If there was no association, the fish in
each test chamber were assumed to be independent of one
another.
If a correlation existed, fish in a test cham-
ber were not considered independent of one another and
the data was analyzed with each test chamber considered
as a replicate.
14
Laboratory Held. Uninfected English Sole:
For use as a comparison in bioassays, gel diffusion
tests and agglutination tests, un infected English sole
were obtained.
Uninfected sole were formalin-disinfected
sole held in the laboratory without trematode infection
for two months.
Serum and Mucus Sample Collection for Bioassays and Immunological Tests:
Fish from which mucus and serum were collected were
anesthetized in a 1:1500 dilution of 2-phenoxyethanol,
rinsed in seawater and drained.
Skin mucus was obtained
by gently scraping the surface of the fish with a glass
slide and collecting the mucus in a petri dish.
Blood
was then collected from the dorsal aorta by severing the
caudal fin or by cardiac puncture.
Mucus was kept on ice
during collection, refrigerated at 4°C overnight, centrifuged for 15 minutes at 1500 x g to remove scales and
other debri and the supernatant collected and stored at 70°C in plastic 5 ml test tubes.
Blood was allowed to
clot for 1-2 hours at room temperature, refrigerated at
4°C overnight, centrifuged for 15 minutes at 1500 x g and
the serum collected and stored at -70°C in microcentrifuge tubes.
Serum and mucus for use in bioassays, gel diffusion
tests and agglutination tests were collected from natur-
15
ally infected juvenile English sole.
Two collections of
sole were made and samples from 25 fish were taken on the
date of capture and then every two weeks following until
no fish remained.
This allowed samples to be collected
from the first group of fish for six weeks and from the
second group of fish for 10 weeks.
Each fish was ex-
amined under a dissecting scope to determine the trematode infection level before sampling.
The serum and
mucus were pooled for each sampling unless there appeared
to be a marked difference in infection levels, then serum
and mucus samples were separated by infection level,
either high or low.
Serum and mucus was also collected from un infected
laboratory held English sole, Buffalo sculpin (Enophrys
bison) collected from Yaquina bay, and chum salmon
(Onchorhynchus
keta) hatched and raised at the Fish
Disease Laboratory.
Gyrodactylus Stellatus Immunized English Sole:
In an effort to obtain English sole serum and mucus
that contained antibodies against
~.
stellatus, 15 for-
malin-disinfected, laboratory held English sole (172.8 ±
23.4 S.E. mm) were anesthetized and injected interperitoneally with 0.1 ml of a 1:1 volume of whole, formalin-killed
(FCA).
~.
stellatus in Fruend's complete adjuvant
Booster injections were administered in the same
16
manner two weeks later.
Four weeks after the booster,
blood was collected by cardiac puncture and mucus was
also collected.
Mucus Bioassays:
Bioassays testing the survival times of
~.
stellatus
in English sole mucus samples were performed in 96-well
polystyrene flat bottomed plates.
performed.
Two bioassays were
Mucus from naturally infected sole held in
the laboratory for varying periods of time, as well as
mucus from uninfected and
compared.
~.
stellatus injected sole were
Buffalo sculpin mucus and seawater were used
as controls.
Bioassays were carried out in a 15°C con-
stant-temperature room.
Live
~.
stellatus were obtained by treating infected
sole with 2-phenoxyethanol for 30 s to 1 min, after which
anesthetized trematodes were removed from the fish surface by a stream of water through a pastuer pipet (Lester
and Adams 1973).
Worms were filtered out of the anes-
thetic on a 53
nitex screen, rinsed in seawater, and
~m
collected in small glass crystallizing dishes.
One to two trematodes were placed in each well of a
96-well plate and brought to a 5% concentration of mucus
in seawater.
mucus.
Control wells contained seawater and no
Seawater used in the tests was from the same
source as the holding water of
~.
stellatus source fish,
17
and varied between 29-32 0/ 00 salinity and 10-16 °C.
Samples were randomly assigned to the rows of the 96-well
plate using a random numbers table, and
plates were tested.
three replicate
Trematodes were monitored every 3-4
hours under a dissecting scope until all worms in the
controls wells had died.
Serum Bioassays:
Bioassays to determine the survival time of
~.
stellatus in English sole serum were conducted in the
same manner as the mucus bioassays.
Serum from two
groups of naturally infected sole held in the laboratory
for varying periods of time, as well as sera from un infected and
~.
stellatus injected sole were compared.
Seawater, buffalo sculpin and chum salmon sera were used
as controls.
Trematode survival was monitored every 0.5-
2 hours until the worms in all serum samples were dead.
Statistical Analysis of Mucus and Serum Bioassays:
Wells in which trematode births occurred were not
analyzed.
The mean and standard error of survival time
were calculated for worms in each treatment replicate.
The assumptions of normality and homogeneity of variance
were tested for with the Shapiro-Wilkes and Bartlett's
test respectively.
When necessary, data were transformed
to meet the assumptions and a one-way analysis of vari-
18
ance CANOVA) was performed.
Dunnett's test was used to
compare the control mean to each of the sample means.
Mucus and serum from newly captured fish were analyzed as
the controls.
In cases of unequal numbers of replicates,
the Bonferroni t-test was used in place of Dunnett's
test.
Student's t-test was used to compare paired sam-
ples.
Preparation of Trematode Antigen for Agglutination and
Gel Diffusion Tests:
Gyrodactylus stellatus for antigen preparation were
obtained by treating infected sole with a 1:4000 dilution
of formalin in seawater for 1 hour.
filtered out of solution on a 53
~m
Trematodes were
nitex screen, rinsed
and preserved in 10% formalin or 70% ethyl alcohol.
For gel precipitin tests, approximately 1 ml of a
packed volume of trematodes was centrifuged for 15 minutes at 1500 xg, washed in PBS three times and homogenized in 2 ml PBS with a Brock tissue grinder.
The BioRad
protein assay was used to determine the concentration of
soluble protein in the supernatant of centrifuged homogenate.
For agglutination tests, the trematode homogenate
was centrifuged and washed three times in PBS to remove
soluble proteins, and the pellet was retained.
concentration of antigen in PBS was used.
A 1% w/v
19
Rabbit Antiserum:
A rabbit antiserum against English sole whole serum
was obtained by injecting a 2-2.5 kg female, New Zealand
White rabbit with a 1:1 volume, 400
English sole whole serum in FCA.
~g/ml
protein, of
The rabbit was injected
with 0.1 ml of antigen intermuscularly in each leg, and
0.1-0.2 ml subcutaneously in five places along the back.
A 10 ml sample of normal rabbit blood was collected by
cardiac puncture before the injections.
A booster of
English sole serum in Freund's incomplete adjuvant, 400
~g/ml
protein, was given two weeks later following the
same injection regime.
Two weeks following the booster
injections, 10 ml of blood was collected from the rabbit
by cardiac puncture.
All injections and bleedings were
performed by trained staff at the O.S.U. Lab Animal Resources facility in Corvallis.
The presence of anti-English sole serum antibodies
in the rabbit antiserum was confirmed in Ouchterlony gel
diffusion tests.
The tests revealed 4-5 precipitation
bands in serum dilutions of 1:1 and 1:2, but none at
higher dilutions.
Gel Diffusion Tests:
An agarose gel, double diffusion precipitation test
(Ouchterlony) was used to detect specific antibodies in
rabbit antiserum, fish serum and fish mucus.
Tests were
20
done in 5.0 cm diameter Gelman plates holding 5 ml of 1%
agarose in .01 M PBS at pH 7.2.
Seven 5 mm diameter
wells placed 10 mm apart (from center to center) were cut
out of the gel, with one well surrounded by six wells.
Each well held approximately 25
~l
of sample.
Following
the addition of the samples, plates were incubated in a
humidity chamber at room temperature and read after 24
and 48 hours (Anderson and Dixon, 1981).
Precipitin tests were performed with normal and immunized rabbit serum against English sole serum; English
sole serum and mucus samples against homogenized
~.
stellatus in PBS; and English sole and buffalo sculpin
mucus samples against rabbit antiserum.
Microtiter Agglutination Test:
A microtiter agglutination test to determine if agglutinating antibodies against
~.
stellatus could be
detected in English sole serum and mucus was performed in
96-well, u-bottom, polystyrene plates following proceedures described by Anderson and Dixon (1981).
Buffalo
sculpin and chum salmon serum samples were tested as controls.
Serum from
~.
stellatus injected English sole was
used as the positive control.
The antigen was a 1% w/v
suspension of homogenized trematodes in PBS.
Samples were assigned to rows on a plate with a
random numbers table.
Each row held eight wells, and two
21
replicate plates were tested.
Two-fold serial dilutions
of a sample in PBS were made in the eight wells (50
per well) of a row.
~l
The first well held undiluted serum
and the eigth held PBS only. Following the serum dilutions, 50
~l
of the antigen suspension was added to each
well of the plate. Plates were gently agitated to mix the
contents of each well, covered and set aside for readings
at 1, 6, and 24 hours.
Macroscopic Slide Agglutination Tests:
Slide agglutination tests to determine if agglutinating antibodies against
~.
stellatus could be
detected in English sole serum and mucus samples were
performed following the proceedures described by Anderson
and Dixon (1981).
Buffalo sculpin, and chum salmon serum
samples were also tested as controls.
Serum from
~.
stellatus injected English sole was used as the positive
control.
The antigen was a 1% wjv suspension of homogen-
ized trematodes in PBS.
One drop of antigen suspension was placed in each
well of an agglutination test slide with a pastuer pipet.
One drop of sample was added to each well on the slide,
one well was left without sample.
Wells were mixed with
wooden applicator sticks and observed for 20 minutes.
22
III.
RESULTS
Gyrodactylus Stellatus on English Sole from Yaguina Bay:
The prevalence and intensity of G. stellatus on 60
juvenile English sole (112 ± 14.6 S.E. mm) collected in
Yaquina Bay in October 1989 was determined and the condition factor of those fish was calculated.
factors averaged 0.98±0.121 S.E ••
Condition
The prevalence of
~.
stellatus was 86.7% and average intensity of infection
was 3.8 ± 3.55 S.E. worms/fish.
The heaviest infection
found was 18.0 worms on one sole.
Handling Stress Experiments:
The influence of handling stress on the infection of
juvenile English sole by
~.
stellatus was tested in three
experiments.
In the first experiment, 80 sole were formalindisinfected, acclimated in laboratory holding tanks for
11 days and test chambers for 15 days.
At test initia-
tion, 40 fish in the treatment group were stressed by
holding them out of water in a dipnet for two minutes, a
procedure repeated three times over the course of an
hour.
Following exposure to the handling stress, all
fish were re-infected with the parasite via two finclipped
~.
stellatus infected sole placed in each test
23
chamber for 72 hours.
The infection intensity levels and
condition factors were determined for the fish that
survived to end of the experiment.
Eighty-five percent
of the unstressed sole survived and only 47% of the
stressed sole survived.
The results of the experiment are presented in Table
1.
Infection intensity levels were 1766.1 ± 1281.63
S.E. worms/fish in the unstressed treatment and
2566.1
±
2004.60 S.E. worms/fish in the stressed treatment (Figure
2).
In the unstressed treatment, ANOVA and regression
analyses showed the number of worms per fish was influenced by the number of fish per test chamber
(0.05~P>0.01,
R=96.7%), with higher infection levels on
fish in tanks with higher densities due to higher survival rates (Figure 1).
This prevented the data from each
fish from being compared individually, requiring instead
analysis of the averaged data from the fish in each test
tank.
There was no significant difference between
stressed and unstressed fish in infection intensity
levels
(0.10~P>0.05)
or condition factors
(0.10~P>0.05).
24
Pigure 1: Regression of the mean number of worms/fish
versus the number of fish per test chamber in the unstressed treatment of the first handling stress experiment.
25
HANDLING STRESS TEST 1
REGRESSION ON UNSTRESSED TREATMENT
50
0
..d
.....
rIl
I%..
45
2
o
~
Q)
p,.
R = 96.7%
(O.05>P>O.Ol)
rIl
S
0
~
0
~
40
~
Q
ItS
Q)
::g
E-<
~
CY
35
CIl
30
~------~--------~--------~------~
4
8
6
#
10
Fish per Test Chamber
. =
95% confidence intervals
Figure 1
12
26
Table 1:
Handling stress experiment 1; the effect of
handling stress on intensity of Gyrodactylus stellatus,
condition factors and survival of laboratory held English
sole after four weeks (1,2).
C-factor±S.E.
(Range)
Treatment
N
surviving N
unstressed
40
34
1766.1±128l.63
(380-6319)
0.90±0.043
(0.72-1.23)
stressed
40
19
2566.l±2004.60
(232-6164)
0.89±0.032
(0.76-1.01)
(0.10<P)
(0.10<P)
Worms/Fish±S.E.
(Range)
Studentt-test
Significance Level
1. Disinfected fish were stressed three times then reinfected.
2. Seawater temperature during the test was 13.9±1.2 °C.
The second handling stress experiment was similiar
to the first except that formalin-disinfected fish were
acclimated longer (holding tanks, 51 days; test chambers,
87 days) and upon test initiation, fish in the stressed
group were held out of water in a dipnet for a singl-e
two-minute treatment.
Thirty-six sole were in the un-
stressed group and 37 sole were in the stressed group.
Survival at the end of the experiment after four weeks
was 100% in both treatments.
Results of the second stress experiment are given in
Table 2.
Infection intensity levels were 926.2 ± 374.73
worms/fish in the unstressed group and 628.2 ± 424.27
worms/fish in the stressed group (Figure 3).
Infection
27
Handling stress experiment 2; the effect of
handling stress on intensity of Gyrodactylus stellatus,
condition factors and survival of laboratory held English
sole after four weeks (1,2).
Table 2:
surviving N
Worms/Fish±S.E.
(Rangel
36
36
926.2±374.73
(312-1924)
1.02±0.072
(0.87-1.18)
37
37
628.2±424.30
(117-2151)
1.00±0.100
(0.85-1.26)
Treatment
N
unstressed
stressed
Student's t-test
Significance Level
(P<0.001)
C-factor±S.E.
(Rangel
(0.10<P)
1. Disinfected fish were stressed once and then reinfected.
2. Seawater temperature during the test was 9.9±1.6 °C.
intensity levels between treatments were significantly
different
(P~0.001)
infections.
with unstressed fish having heavier
Fish condition factors were not significant-
ly different between treatments
(0.10~P).
The third handling stress experiment was again
similiar except that test fish were not formalin-treated
and reinfected but rather carried a low level of infection that was observed to be typical for fish that survived a laboratory epizootic
(~300
worms/fish).
General
infection intensity levels at the beginning of the test
were estimated by examining two sole under a dissecting
scope.
Sole were held in holding tanks for 6 months and
acclimated to test chambers for 6 weeks.
The unstressed
28
treatment had 12 sole and the stressed treatment had 13
sole.
Ninety-two percent of the unstressed sole survived
and 85'% of the stressed sole survived four weeks to the
end of the experiment.
Results of the third handling stress experiment are
presented in Table 3.
Infection intensity levels were·
292.9 ± 315.90 S.E. worms/fish in the unstressed treatment and 1637.9 ± 2673.20 S.E. worms/fish in the stressed
treatment (Figure 4).
Sole in the stressed treatment had
significantly higher infection levels than sole in the
unstressed treatment
(0.01<P~0.05).
Condition factors
between treatments were not significantly different
(O.OI~P).
Table 3: Handling stress experiment 3; the effect of
handling stress on intensity of Gyrodactylus stellatus,
condition factors and survival of laboratory held English
sole after four weeks (1,2).
Surviving N
Worms/Fish±S.E.
(Rangel
C-factor±S.E.
(Rangel
Treatment
N
unstressed
12
11
292.9±315.90
(70-1016)
0.92±0.077
(0.83-1.02)
Stressed
13
11
1637.9±2673.20
(123-9249)
0.92±0.059
(0.79-0.99)
Student's t-test
Significance Level
{0.05>P>0.01)
{0.10>P)
1. Test fish were survivors of a laboratory epizootic,
not disinfected and were stressed three times.
2. Seawater temperature during the test was 13.1±1.5 °C.
29
Piqure 2: Handling stress experiment 1; the effect of
handling stress on intensity of Gyrodactylus stellatus on
laboratory held English sole after four weeks. Formal indisinfected fish were stressed by holding them out of
water in a dipnet for two-minutes three separate times
over the course of an hour. Fish were then re-infected
via two fin-clipped ~. stellatus infected sole. Infection intensity levels of the two treatments were not
significantly different (Student's t-test, O.10>P).
Piqure 3: Handling stress experiment 2; the effect of
handling stress on intensity of Gyrodactylus stellatus on
laboratory held English sole after four weeks. Formal indisinfected fish were stressed by holding them out of
water in a dipnet for two-minutes. Fish were then reinfected via two fin-clipped ~. stellatus infected sole.
Infection intensity levels between the two treatments
were significantly different (Student's t-test, P50.001).
Piqure 4: Handling stress experiment 3; the effect of
handling stress on intensity of Gyrodactylus stellatus on
laboratory held English sole after four weeks. Sole
maintained low infection intensities from a previous
laboratory epizootic. Test fish were stressed by
holding them out of water in a dipnet for two-minutes
three separate times over the course of an hour. Infection intensity levels between the two treatments were
significantly different (Student's t-test, O.05~P>O.Ol).
HANDLING STRESS TEST 1
3000
...c=
I/)
.~
~
""'-.
til
£3
1-0
2500
2000
0
~
1500
~
1000
~
Id
Q)
::a
500
0
1
Unstressed
~
Figure 2
HANDLING STRESS TEST 2
3000
...c=
.~
2
Stressed
2500
E 2000
""'-.
1-0
~
1500
~
1000
~
Id
Q)
:::!:1
500
o '----
1
Unstressed
...c=
til
~
3000
Figure 3
2
Stressed
HANDLING STRESS TEST 3
~---~---------~---~
2500
E 2000
""'-.
1-0
~
1500
~
1000
~
Id
Q)
:::!:1
500
o '----
1
Unstressed
2
Figure 4
Stressed
30
31
To determine how infection intensity levels and
condition factors of sole used in the handling stress
experiments compared to sole in Yaquina bay, infection
intensity levels and condition factors were compared.
Infection intensity levels on fish in all of the handling
stress experiments were significantly higher than infection intensity levels of newly captured sole (Student's
t-test,
P~O.OOl).
Condition factors were not signifi-
cantly different with the exception of the condition
factors of the stressed treatment in the second handling
stress experiment which were significantly higher than
those of newly captured fish
(0.05~P>0.01)
(see page 22).
Substrate Experiment:
The influence of substrate on
~.
stellatus infection
levels on laboratory held English sole was tested by
holding seventy-nine (95.5 ± 9.25 S.E. mm) newly captured, naturally infected sole in tanks with or without a
sand substrate for two weeks.
Forty sole were placed in
tanks without substrate and 39 sole were placed in tanks
with substrate.
Survival at the end of the experiment
was 100% in both treatments.
Results are given in Table 4.
Infection intensity
levels were 54.9 ± 32.44 S.E. worms/fish on the fish in
tanks without substrate and 37.3 ± 21.97 S.E. worms/fish
on the fish in tanks with substrate (Figure 5).
Infec-
32
tion intensity levels and condition factors were both
significantly different between treatments, with fish in
tanks with substrate having higher condition factors
(P<0.001) and lower infection rates
(P~0.001)
than fish
in tanks without substrate.
To determine how infection intensity levels and
condition factors of sole used in the substrate experiment compared to sole in Yaquina bay, infection intensity
levels and condition factors were compared.
Infection
intensity levels were significantly higher than infection
intensity levels of newly captured sole
(P~0.001)
and
condition factors were significantly lower than those of
newly captured sole
(P~0.001)
(see page 22).
Table 4: The effect of substrate on intensity of
Gyrodactylus stellatus, condition factors and survival of
laboratory held English sole after two weeks (1,2).
Treatment
N
surviving N
No Substrate
40
40
54.9±32.44
(10-179)
0.77±0.046
(0.69-0.92)
Substrate
39
39
37. 3±21. 97
(5-106)
0.86±0.074
(0.73-1.09)
(P<O.OO1)
(P<O.OOll
Student's t-test
Significance Level
Worms/Fish±S.E.
(Rangel
C-factor±S.E.
(Rangel
1. Test fish were newly captured, naturally infected and
held in test chambers with or without a sand substrate.
2. Seawater temperature during the test was 12.9±0.8 °C.
33
The effect of substrate on infection intensity
levels of Gyrodactylus stellatus on laboratory held
English sole after two weeks. Infection intensity levels
between the two treatments were significantly different
(Student's t-test, P~O.OOl).
Figure 5:
34
100
SUBSTRATE TEST
~------~------------------~------~
80
..Q
.....rIl
r:..
"-..
e....
60
d
40
rIl
0
~
=II::
td
::a
OJ
20
o
L - -_ _
Without Substrate
Figure 5
2
With Substrate
35
starvation ExPeriment:
To determine if
~.
stellatus had an effect on the
survival of unfed English sole, the survival times of
unfed sole that were naturally infected with the parasite
were compared to the survival times of disinfected, unfed
sole.
Each treatment had 40 fish and dead fish were
observed under a dissecting scope to confirm the presence
or absence of worms depending on the treatment.
Worms
were found to be present on fish in one test chamber that
contained disinfected fish, data from those fish were not
included in statistical analyses.
Results of the starvation experiment are presented
in Table 5.
The mean survival time of the infected sole
was 77.5 ± 7.37 S.E. days and mean survival time of the
disinfected sole was 136.5 ±
Mean survival times
(P~0.001)
(P~0.001)
34~58
S.E. days (Figure 6).
and condition factors
were significantly different between infected
and disinfected fish, with disinfected fish living longer
and having higher condition factors.
To determine how condition factors of sole used in
the starvation experiment compared to sole in Yaquina
bay, condition factors were compared.
Condition factors
of fish in the experiment were significantly lower than
condition factors of newly captured sole
(P~0.001).
36
TABLE 5: The effect of Gyrodactylus stellatus on survival time and condition factors of unfed, newly captured, naturally infected and disinfected English sole
held in test chambers until death (1).
Days Survival±
S.E. (Rangel
C-factor±S.E.
(Rangel
Treatment
N
Infected
40
0.77)
Disinfected
77.5±7.37
(58-97)
0.62±0.070
(0.51-
28
136.5±34.58
(75-199)
0.50±0.072
(0.36-
0.64)
Student's t-test
Significance Level
(P<O.OOll
(P<O.OOll
1. Seawater temperature during the test was 12.3±8.9
°c.
37
Figure 6: The effect of Gyrodactylus stellatus on survival time of unfed, newly captured, naturally infected
and disinfected English sole held in test chambers until
death. Mean survival times between the two treatments
were significantly different (Student's t-test, P~O.OOl).
38
200
"t:I
STARVATION TEST
~------~------------------~-------.
150
Cl)
.....:>:>
""
~
CIl
CIl
>..
ro 100
Q
~
d
«I
Cl)
~
50
o
L - -_ _
2
Disinfected
Infected
Figure 6
39
Mucus Bioassays:
To assertain if English sole mucus contains components that are involved in resistance to monogenetic
trematode infection, two bioassays testing the survival
times of
~.
stellatus in English sole mucus collected
from two groups of fish at different times during a
laboratory infection were performed.
In both bioassays,
mucus from the newly captured fish in each group served
as the treatment to which all other treatments were
compared.
In the first bioassay, samples were collected from a
group of sole at capture and every two weeks for six
weeks.
At six weeks, mucus samples collected from sole
that carried heavy infections were tested separately from
those collected from sole that carried light infections.
In heavy infections, trematodes numbered in the thousands
with the sole bearing dense patches of trematodes on the
fins; in light infections, trematodes numbered in the
hundreds and were more evenly and widely distributed over
the fins of the sole than in heavy infections.
Buffalo
sculpin mucus and seawater served as negative controls.
In the first mucus bioassay test, trematodes survived significantly longer in seawater (20.5±4.42 S.E.
hours) than they did in mucus from newly captured sole
(8.7±1.45 S.E. hours) (Dunnett's test, P50.05).
There
was no significant difference between trematode survival
40
time in mucus from newly captured sole and any other
mucus sample (Table 6 and Figure 7).
Trematode survival
time in mucus collected from heavily infected fish after
six weeks (9.0±1.31 S.E. hours) was significantly longer
than in mucus collected from lightly infected fish after
six weeks (5.2±0.89 S.E. hours) (Student's t-test,
P50.001).
Table 6: Mucus bioassay 1; the effect of English sole
mucus on the mean survival time (MST) of Gyrodactylus
stellatus. Mucus was collected from sole upon capture
and every two weeks following for six weeks. Samples
taken at six weeks were separated by infection level
(heavy and light). Controls were Buffalo sculpin mucus
and seawater.
Sample
N
# of
Replicates
MST±S •E. (hours)
(Rangel
Newly captured
43
3
8.7±1.45
(7.3-10.2)
2 Weeks
49
3
11.0±1.12
(9.87-12.11)
4 Weeks
46
3
10.7±1.15
(4.4-6.1)
6 Weeks Heavy
49
3
9.0±1.31
(7.9-10.4)
6 Weeks light
42
3
5.2±0.89
(4.4-6.1)
Buffalo Sculpin
49
3
11.1±2.39
(8~6-13.3)
Seawater*
39
3
20.5±4.42
(15.7-24.3)
* Denotes samples significantly different from newly
captured (Dunnett's test, P50.05).
41
Piqure 7: Mucus bioassay 1; the effect of English sole
mucus on the mean survival time of Gyrodactylus
stellatus. Mucus was collected from sole upon capture
and every two weeks following for six weeks. Samples
taken at six weeks were separated by infection level
(heavy and light). Controls were Buffalo sculpin mucus
and seawater.
42
25
...........
Ul
MUCUS BIOASSAY 1
~-~--~----~--~-----r---~----~--~~
*
20
'"'
::l
0
..q
.........
15
Q)
.....S
E-t
ro
-"'
.....>
>
10
'"'
rn
::l
Q
ro
Q)
::iil
5
o
2
3
4
6
5
7
8
Treatment
*=
II
D
~
~
Significantly different from newly captured
OJ = 6 Weeks Light (5)
=
Newly Captured (1)
=
2 Weeks (2)
=
4 Weeks (3)
EE
=
=
6 Weeks Heavy (4)
~
= Seawaler (8)
EJ =
Figure 7
Uninfecled (6)
Buffalo Sculpin (7)
43
In the second bioassay, mucus samples were collected
from a group of sole at capture and every two weeks
following for ten weeks.
Mucus samples collected from
heavily infected fish after eight weeks and 10 weeks were
tested separately from those collected from lightly
infected fish.
Survival of
stellatus was also tested
~.
in mucus from un infected and
~.
stellatus injected sole.
Survival of worms in Buffalo sculpin mucus and in seawater served as controls.
Trematode survival times in mucus from lightly infected (recovering) sole at eight weeks (10.2 ± 0.071
S.E. hours) and ten weeks (9.3 ± 0.94 S.E. hours) were
significantly lower than was trematode survival time in
mucus from newly captured sole (15.6 ± 3.40 S.E. hours)
(Bonferroni t-test,
P~0.05).
Trematode survival in
seawater (26.0 ± 4.41 S.E. hours) was significantly
higher than trematode survival in mucus from newly captured sole (Figure 8).
The survival
of~.
stellatus in
all other mucus samples was not significantly different
from that in mucus from newly captured sole.
Results
from the second mucus bioassay are presented in Table 7.
Trematode survival time in mucus from lightly infected
sole was significantly shorter than that in mucus collected from heavily infected sole in both the eight and
10 weeks samples (Student's t-test,
P~0.001).
44
Table 7: Mucus bioassay 2; the effect of English sole
mucus on the mean survival time (MST) of Gyrodactylus
stellatus. Mucus was collected from sole upon capture
and every two weeks following for ten weeks. Samples
taken at eight and ten weeks were separated by infection
level (heavy and light). Mucus samples from uninfected
and immunized sole were also tested. Controls were
Buffalo sculpin mucus and seawater.
MST±S . E. (hours)
(Rangel
Sample
N
# of
Replicates
Newly captured
20
3
15.6±3.40
(11.0-18.6)
2 Weeks
24
3
14.4±0.88
(13.7-15.4)
4 Weeks
28
3
16.6±1.03
(15.5-17.6)
6 Weeks
24
3
14.2±2.76
(12.4-17.4)
8 Weeks Heavy
21
2
15.2±0.04
(15.2-15.2)
8 Weeks Light*
31
3
10.2±0.71
(9.5-10.9)
10 Weeks Heavy
25
3
13.4±2.33
(10.7-15.0)
10 Weeks Light*
31
3
9.3±0.94
(8.2-10.0)
Uninfected
28
3
18.8±0.86
(18.3-19.8)
Immunized
31
3
14.05±2.53
(11.6-16.6)
Buffalo Sculpin
28
3
18.7±0.84
(17.9-19.6)
Seawater*
22
4
26.0±4.41
(20.7-30.6)
* Denotes samples significantly different from newly captured (Dunnett's test, P~0.05).
45
Figure 8: Mucus bioassay 2; the effect of English sole
mucus on the mean survival time of Gyrodactylus
stellatus. Mucus was collected from sole upon capture
and every two weeks following for ten weeks. Samples
taken at eight and ten weeks were separated by infection
level (heavy and light). Mucus samples from uninfected
and immunized sole were also tested. Controls were
Buffalo sculpin mucus and seawater.
46
MUCUS BIOASSAY 2
35
*
30
,-...
CIl
~
~
0
,.q
..........
QJ
.....E!
25
20
E-o
~
Id
.....>
t~
15
U)
Q
Id
10
QJ
:::i!I
5
o
2
3
4
5
6
7
8
9
10
11
12
Treatment
*= Significantly different from
II =
D=
~
~
Newly Captured (1)
2 Weeks (2)
= 4 Weeks (3)
= 6 Weeks (4)
OJ = 8 Weeks Heavy (5)
B = 8 Weeks Light (6)
§]
~
newly captured
= 10 Weeks Heavy (7)
= 10 Weeks Light (8)
I = Uninfected (9)
illll = Immunized (10)
~
I
Figure 8
= Buffalo Sculpin (11)
= Seawater (12)
47
Serum Bioassays:
To assertain if English sole serum contained factors
involved in resistance to trematodes, two bioassays
testing the survival times of
~.
stellatus in English
sole serum collected from two groups of fish at different
times during a laboratory infection were performed.
Serum samples were collected at the same time as were
mucus samples for the mucus bioassay tests.
The only
serum samples that were tested were those from fish whose
mucus produced a bioassay result suggesting the presence
of a factor that affected
~.
stellatus survival.
In both
bioassays, serum from the newly captured fish in each
group served as the treatment to which all other treatments were compared.
In the first bioassay, serum samples collected from
a group of sole upon capture and after two weeks were
tested.
In the second bioassay, serum samples collected
from a group of sole upon capture, after eight weeks and
after 10 weeks were tested.
The eight and ten week
samples were separated according to trematode infection
intensity as described above.
~.
Sera from uninfected and
stellatus injected sole were also tested.
Buffalo
sculpin and chum salmon sera served as controls.
In the first group of sole, trematode survival times
in the two week and uninfected sole sera did not differ
significantly from newly captured sole.
Trematode
48
survival times in buffalo sculpin serum (11.0 ± 1.21 S.E.
hours) was significantly higher than survival in serum
from newly captured sole (5.3 + 0.37 S.E. hours)
(Bonferroni t-test,
P~0.05).
Results of the serum bioas-
say using serum from the first group of sole are given in
Table 8 and Figure 9.
Serum bioassay 1; the effect of English sole
serum on the mean survival time (MST) of Gyrodactylus
stellatus. Serum was collected from sole upon capture
and after two weeks. Serum from immunized sole was also
tested. Controls were Buffalo sculpin and chum salmon
sera.
Table 8:
# of
Replicates
MST±S •E. (hours)
(Range)
Sample
N
Newly captured
24
3
5.3±0.37
(4.9-5.6)
2 Weeks
22
3
5.0±0.33
(4.6-5.3)
Uninfected
11
2
5.4±0.23
(5.3-5.6)
Immunized**
17
2
3.0±0.00
Buffalo Sculpin*
20
3
11. 0±1. 21
(9.8-12.3)
Chum Salmon**
28
3
0.5±0.00
* Denotes samples significantly different from newly
captured (Dunnett's test, P~0.05).
** The treatments couldn't be compared statisically
because there was no variance.
49
Figure 9:
Serum bioassay 1; the effect of English sole
serum on the mean survival time of Gyrodactylus
stellatus. Serum was collected from sole upon capture
and after two weeks. Serum from immunized sole was also
tested. Controls were Buffalo sculpin and chum salmon
sera.
50
Serum Bioassay 1
14
*
12
........
rn
s...
~
0
,Q
10
'-'"
....e
8
....>>
6
Q)
E-o
......
ttl
s...
~
rt.l
Q
ttl
Q)
4
::?!!
2
o
4
3
2
5
6
Treatment
* = Significantly different from newly captured
II
= Newly Captured (1)
D=
~
2 Weeks (2)
= Uninfected (3)
~
OJ
B
Figure 9
= Immunized (4)
= Buffalo Sculpin (5)
= Chum Salmon (6)
51
When serum samples from the second group of sole
were tested, trematode survival times in all serum samples, with the exception of uninfected sole, were significantly different from survival times in newly captured sole serum (Bonferroni t-test,
P~0.05).
Trematode
survival times in sera from lightly infected sole at
eight weeks (5.0 ± 0.29 S.E. hours) and heavily (4.5 ±
1.12 S.E. hours) and lightly (1.7 ± 0.16 S.E. hours)
infected sole at ten weeks were significantly shorter
than in newly captured sole serum (6.8 ± 1.08 S.E.
hours).
Trematode survival time in sera from buffalo
sculpin (11.0
± 1.21 S.E. hours) and heavily infected
sole at eight weeks (11.5
± 0.73 S.E. hours) were sig-
nificantly higher than in serum from newly captured sole.
Results of the serum bioassay using serum from the second
group of sole are given in Table 9 and Figure 10.
Trema-
tode survival time in serum collected from lightly infected sole was significantly shorter than that in serum
collected from heavily infected sole in both the eight
and 10 weeks samples (Student's t-test,
P~O.OOl).
52
Table 9: Serum bioassay 2; the effect of English sole
serum on the mean survival time (MST) of Gyrodactylus
stellatus. Serum tested was collected from sole upon
capture and at eight weeks and 10 weeks. Eight and ten
week samples were separated by infection levels (heavy
and light). Serum from immunized sole was also tested.
Controls were Buffalo sculpin and chum salmon sera.
41 of
Replicates
MST±S • E. (hours)
(Rangel
Sample
N
Newly captured
26
3
6.8±1.08
(5.7-7.9)
8 Weeks Heavy*
19
3
11.5±0.73
(10.7-12.0)
8 Weeks Light
22
3
5.0±0.29
(4.7-5.3)
10 Weeks Heavy*
27
3
4.5±1.12
(3.6-5.8)
10 Weeks Light*
25
3
2.7±0.16
(2.6-2.9)
Uninfected
11
2
5.4±0.23
(5.3-5.6)
Immunized**
17
2
3.0±0.00
Buffalo Sculpin*
20
3
11.0±1.21
(9.8-12.3)
Chum Salmon**
28
3
O.5±O.OO
* Denotes samples significantly different from newly
captured (Dunnett's test, P.$.0.05).
** The treatment couldn't be compared statistically
because there was no variance.
53
Fiqure 10: Serum bioassay 2; the effect of English sole
serum on the mean survival time of Gyrodactylus
stellatus. Serum tested was collected from sole upon
capture and at eight weeks and 10 weeks. Eight and ten
week samples were separated by infection levels (heavy
and light). Serum from immunized sole was also tested.
Controls were Buffalo sculpin and chum salmon sera.
54
Serum Bioassay 2
14
12
*
*
....-rn
r....
::J
0
..d
........
10
Q)
....E-<El
-....
8
ttl
po
po
r....
::J
6
rf.l
d
4
ttl
Q)
::a
2
o
2
3
4
5
7
6
8
9
Treatment
II
* = Significantly different from newly captured
= Newly Captured (1)
D=
8 Weeks Heavy (2)
~
~
= 8 Weeks Light (3)
=
10 Weeks Heavy (4)
OJ = 10 Weeks Light (5)
§
~
~
~
Figure 10
= Uninfected (6)
=
Immunized (7)
= Buffalo Sculpin (8)
=
Chum Salmon (9)
55
Gel Diffusion. Microtiter and Slide Agglutination Tests:
In an effort to determine if serum from English sole
infected with
~.
stellatus contained precipitins (e.g.
antibodies) against
~.
stellatus, gel diffusions of serum
and mucus from infected sole were run against homogenized
worms.
No evidence of the presence of precipitins was
found, possibly due to an inadequate (31
~g/ml)
amount of
soluble protein in the worm preparation.
Microtiter and slide agglutination tests to determine if agglutinating antibodies against
~.
stellatus
could be detected in serum and mucus from trematode infected sole were attempted.
Agglutinating antibodies
were not detected by either method.
An effort was then made to determine if the mucus
from~.
stellatus infected English sole contained factors
also found in the serum of infected sole.
This was de-
termined by Ouchterlony tests in which mucus samples were
diffused with rabbit antiserum against English sole
serum.
Rabbit antiserum was in the center well of the gel
with undiluted samples of mucus described in the mucus
bioassays in the surrounding wells.
Sera from buffalo
sculpin and chum salmon served as controls.
English sole
serum served as a positive control.
The rabbit antiserum recognized serum factors in
most of the English sole mucus samples, with differences
56
in the strength of the precipitation reaction and/orthe
number of precipitating bands (Figures 11, 12, and 13).
stronger precipitation reactions occurred in mucus samples collected at later times during the trematode infection.
The rabbit antiserum did not recognize any serum
factors in mucus from
~.
stellatus injected sole, unin-
fected sole, and buffalo sculpin and chum salmon sera.
The rabbit antiserum formed four to five precipitating
bands with the positive control, English sole serum.
Results of the fish mucus, rabbit antiserum Ouchterlonys
are given in Table 10.
To test for the effect of prozone, an Ouchterlony
test was done with dilutions of 1:1, 1:2, 1:3, 1:4, 1:5
and 1:50 of mucus in PBS from uninfected sole in the
surrounding wells and undiluted rabbit antiserum in the·
center well.
As a control, mucus from lightly infected
sole at 10 weeks was diluted in the same manner on a
second Ouchterlony plate.
No precipitation bands were
seen in any of the dilutions of the mucus from uninfected
sole, and a single band of precipitation was present in
all dilutions except 1:50 in the mucus from lightly
infected sole at 10 weeks.
These results indicate that
prozone was not inhibiting precipitation band formation,
confirming that the mucus from uninfected sole did not
contain factors antigenically related to those in English
sole serum.
57
Table 10: Results of Ouchterlony tests of English sole
mucus samples collected from two groups of sole (1)
diffused with rabbit antiserum against English sole
serum. Mucus from uninfected and immunized sole, and
serum from buffalo sculpin and chum salmon were also
tested. English sole serum served as a positive control.
Sample
# of Precipitation bands
Precipitation Relative
to Newly Captured
Group 1:
Newly captured
2 Weeks
4 Weeks
6 Weeks Heavy
6 Weeks Light
1
1
1
1
2-3
Weaker
Same
Same
Stronger
Group 2:
Newly captured
2 Weeks
4 Weeks
6 Weeks
8 Weeks Heavy
8 Weeks Light
10 Weeks Heavy
10 Weeks Light
1
1
1
1
4-5
1
4-5
1
Weaker
Weaker
Same
Stronger
Stronger
Stronger
Stronger
Uninfected sole
Immunized sole
o
o
Sera:
English Sole
Buffalo Sculpin
Chum Salmon
4-5
o
Stronger
o
1. Mucus was collected upon capture, and every two weeks
following. Samples were separated by infection
level (heavy or light) in some samples.
58
11: Plate 1; Ouchterlony test to detect English
sole serum factors in English sole mucus. Mucus was
collected from the first group of sole upon capture and
every two weeks following for six weeks. Samples taken
at six weeks were separated by infection level (heavy and
light). The center well contained rabbit antiserum
against English sole serum. The first well contained
English sole serum and served as a positive control (1).
The remaining wells contained English sole mucus from:
uninfected (2); newly captured (3); two weeks (4); four
weeks (5); and six weeks (heavy infection) (6).
~igur.
Plate 2; Ouchterlony test to detect English
sole serum factors in English sole mucus. Mucus was collected from two groups of sole upon capture and every two
weeks following. Samples were separated by infection
level (heavy and light). The center well contained
rabbit antiserum against English sole serum. The center
well contained English sole serum and served as a positive control (1). The remaining wells contained English
sole mucus from: six weeks (light infection), group 1
(2); and newly captured (3), two weeks (4), four weeks
(5), and six weeks (6) from group 2 sole.
Figure 12:
59
I'iqure 11
Fiqure 12
60
Plate 3; Ouchterlony test to detect English
sole serum factors in English sole mucus. Mucus was
collected from the second group of sole upon capture and
every two weeks following for ten weeks. Samples were
separated by infection level (heavy and light). The
center well contained rabbit antiserum against English
sole serum. The first well contained English sole serum
and served as a positive control (1). The remaining
wells contained English sole mucus from: immunized (2);
eight weeks (heavy infection) (3); eight weeks (light in~
fection) (4); ten weeks (heavy infection) (5); and ten
weeks (light infection) (6).
Piqure 13:
61
Fiqure 13
62
IV.
DISCUSSIOB
The purpose of this study was to explore the mechanisms of English sole resistance to
~.
stellatus by ex-
amining the basis for the transitory loss of resistance
in the laboratory reported by Kamiso and Olson (1986).
Previous studies determined that although water temperature, nutrition, and crowding were factors that influenced the host-parasite relationship between juvenile
English sole and
~.
stellatus, they did not account for
the high infection intensities that develop on laboratory
held English sole (Handoyo 1983).
stresses associated
with handling, transport, and captivity are known to
influence the disease susceptibility of fishes by suppressing immune responses (E11saesser and Clem, 1986;
Miller and Tripp, 1982) and may explain changes in English sole resistance to
~.
stellatus.
The three handling stress experiments in this study
differed in length of acclimation, method of infection,
and the extent of handling stress to which test fish were
exposed to.
In the first experiment, there was no dif-
ference in the infection intensity levels between treatments; in the second experiment, fish in the stressed
treatment had significantly fewer trematodes than did
fish in the unstressed treatment; and in the third ex-
63
periment, fish in the unstressed treatment had significantly fewer worms than fish in the stressed treatment.
Khalil (1964) found that Polypterus seneqalus that
recovered from infections by the Macrogyrodactylus
polypteri were not susceptible to reinfection so long as
they retained a few trematodes.
If the fish remained
without the parasite for a 'short while', reinfection was
possible.
Lester and Adams (1974b) observed that
threespine sticklebacks that had lost their
~.
alexanderi
infections were refractory to further infections for
about three weeks and then were susceptible to reinfection.
In the first two handling stress experiments, the
infection intensity levels of the unstressed (control)
treatments, were high.
One explanation for this may be
that the sole had lost resistance to the parasite during
their trematode-free, pre-test acclimation period which
exceeded three weeks in both experiments.
In the second experiment, trematode infection intensities were higher on the unstressed fish than on the
stressed fish.
The level of stress exposure in the
second experiment was lower than the level of stress in
the first and third experiments, and may not have been
sufficient enough to cause a response.
Fish in the
second experiment were observed to secrete large quantities of mucus during exposure to the handling stress.
64
This in combination with a loss of trematode resistance
in both the stressed and unstressed groups may have given
the stressed fish a slight advantage over the unstressed
fish when the parasite was reintroduced
and could have resulted in lower infection levels on the
fish that received the handling stress.
The third handling stress experiment was probably
the most accurate re-creation of
~.
stellatus infection
dynamics that occur when fish are captured and brought to
the laboratory.
through a
~.
The fish were not disinfected, went
stellatus epizootic, recovered and had
relatively low infection intensity levels at test initiation seven months after capture.
Fish in the treatment
group were exposed to the same handling stress as were
fish in the first experiment, and infection intensity
levels of stressed fish were significantly higher than
those of unstressed individuals.
Infection intensity
levels of the unstressed group (control) remained close
to the low levels typical for fish that had survived a
laboratory epizootic (Kamiso and Olson 1986).
The results suggest that handling stress can influence
the host-parasite relationship between English sole and
~.
stellatus, and that stresses associated with capture
and laboratory holding may be the cause of the increased
trematode intensity levels in laboratory held fish.
Results of the first two experiments were probably in-
65
fluenced by manipulations that masked attempts to experimentally emulate handling stress.
In the estuary, juvenile English sole are found in
areas with sand or mud substrate (Toole et ale 1987).
In
the experiment designed to assess the influence of substrate on
~.
stellatus infections of laboratory held
English sole, fish in the tanks with substrate had significantly fewer worms than the fish in tanks without
substrate.
During the experiment, the fish in the tanks
with substrate were observed to bury themselves, startle
less easily and eat more readily than fish in tanks
without substrate.
Although the reduced number of trematodes on fish in
the substrate treatment may have been due to mechanical
effects of the sand, the behavior of the fish in this
treatment suggested they may have also been under less
stress than fish in tanks without substrate.
The dif-
ference in infection intensity levels between treatments
was possibly due to reduced stress and accompanying
nutritional advantages in the group with substrate.
A previous study reported a higher rate of
~.
stellatus increase on unfed English sole than on fed sole
and also that fed fish dying with heavy trematode infections ceased feeding and became emaciated before succumbing (Kamiso and Olson, 1986).
They suggested that death
may have been due to the combined effects of starvation
66
and heavy parasitism, but they did not separate the
effects of starvation and trematode infection on mortality.
An
experiment designed to separate the effect of
~.
stellatus infection and starvation on mortality showed
that the survival times of unfed, trematode infected sole
were significantly less than survival times of unfed,
disinfected sole so
~.
stellatus infection does ac-
celerate the rate at which unfed fish die.
Humoral antibody responses appear to be the only
mechanism yet described for helminth mediated immunity in
fishes (Evans and Gratzek, 1989).
Precipitating, IgM-
like antibodies against digenetic (Cottrell 1977) and
acanthocephalan (Harris 1972) parasites have been detected in the sera of infected fish.
Nigrelli (193Sa) studied the effects of marine fish
mucus on the monogenetic trematode
~.
melleni and found
that mucus from fish with natural immunity to the parasite shortened trematode survival under experimental
conditions.
Hanson (1973) observed similiar results when
he exposed the monogenetic trematode
~.
embiotoci to
serum and mucus from the striped surfperch (Embiotoca
lateralis), a fish with natural resistance to the parasite.
He suggested that specific antibodies in the mucus
were involved in the resistance based on results of
hemagglutination tests.
The exact mechanisms operating
67
in resistance to monogenetic trematode infections is not
known, but it has been suggested that resistance in
gyrodactylid infections may be associated with mucus
secretions (Evans and Gratzek, 1989: Scott and Robinson,
1984; Lester and Adams, 1974a: Handoyo 1983).
The mucus bioassays performed in this study indicated that both the mucus and serum of English sole were
involved in resistance to the
~.
stellatus.
Generally,
trematode survival was shorter in mucus and serum samples
collected at later times in the laboratory infection,
when sole were beginning to show signs of recovery; and
trematode survival time in the mucus and serum from
lightly infected (recovering) sole was always significantly shorter than trematode survival time in the mucus
from heavily infected sole held in the laboratory for the
same period of time.
These results suggested that the
factors causing resistance in the mucus of English sole
were also present in the serum, and that these factors
may result in recovery from the infection.
Results of
the gel diffusion tests gave evidence to support this.
Proteins antigenically related to serum factors have
been detected in the mucus of bass, catfish, and rainbow
trout (O'rourke, 1961; Di Conza and Halliday, 1971;
Harrell et al., 1976).
In this study, rabbit antiserum
against English sole serum was used in gel diffusion
tests to determine that factors in English sole skin
68
mucus were antigenically related to factors in English
sole serum, but these factors were not characterized.
These factors were detected in mucus collected from sole
at all times during a laboratory infection.
Generally,
bands of precipitation that indicated antigenic recognition were weakest in mucus collected from sole during
periods of increasing infection intensity, and were
strongest in mucus from sole at later stages of infection.
No precipitation reactions were detected in the
mucus of uninfected sole, indicating that the precipitation bands that were observed were associated with
~.
stellatus infections.
Multiple serum factors were detected in Ouchterlony
tests on mucus from sole still heavily infected at late
stages of infection (eight and ten weeks), and a single
band was detected in Ouchterlony tests on mucus from
lightly infected sole at late stages of infection.
The
factor detected in mucus from the lightly infected sole
may be associated with resistance to the parasite because
the serum and mucus of lightly infected sole had the
greatest effect on trematode survival times, and it was
from fish that were recovering from the infection.
The reason for the differences in the number of
precipitation bands detected in mucus at heavy and light
levels of infection is not known.
be leakage of
One explanation could
serum into the mucus of heavily infected
69
fish through small areas of hemorrhaging on the fins of
heavily infected sole.
Di Conza and Halliday (1971) identified a protein in
the mucus of catfish which shared antigenic determinants
with catfish serum antibody, they determined that this
protein was immunoglobulin, but because it did not have
antibody activity found at the same time as in serum,
they suggested that the immunoglobulin was locally synthesized rather than derived from the blood.
In con-
trast, the mucus of plaice was shown to contain antibodies similiar to those found in serum (Fletcher and
Grant, 1969).
Although the results of the gel diffusion
tests in this study do not allow us to conclude that the
factors in the serum and mucus are identical it is likely
that they are for the following reasons:
The serum and
mucus of English sole had similiar effects on trematode
survival times; and gel diffusion tests detected factors
in the mucus antigenically similiar to factors in the
serum.
Lester (1972) found that intramuscular injections of
whole
~.
alexanderi antigen conferred no protection in to
threespine sticklebacks and Nigrelli (1935b) observed
similiar results when pompano (Trachinotus carolinus)
were injected with ground, dried and fresh
Immunizing English sole with homogenized
~.
E. melleni.
stellatus in
FCA failed to illicit an antibody response detectable by
70
the methods used in this study.
Neither precipitating or
agglutinating antibodies were detected; but, although the
results couldn't be analyzed statistically, serum from
immunized sole did appear to have an effect on trematode
survival times (Figures 9 and 10).
Because of problems
with the methodology, the failure to detect
~.
stellatus
antibodies in English sole can not be interpreted to mean
that antibodies weren't produced.
Low levels of soluble
protein in the trematode antigen preparation may be the
reason for negative reactions in the gel diffusion tests,
and neither the gel diffusion nor agglutination tests had
a positive control, such as rabbit antiserum against
~.
stellatus.
In summary, the infection intensity of
~.
stellatus
on laboratory held English sole was influenced by handling stress, substrate, and starvation.
Trematode sar-
vival times were significantly reduced in the serum and
mucus samples collected from sole at the later, recovering stages of infection, suggesting that both the serum
and mucus of English sole are involved in resistance.
The mucus of
~.
stellatus infected English sole contained
factors antigenically similiar to factors in English sole
serum.
These factors were not present in the mucus of
uninfected sole.
Precipitation bands in the gel dif-
fusions tests appeared to be the strongest in mucus
samples from sole at later, recovering stages of infec-
71
tion.
Results of the serum and mucus bioassays and the
Ouchterlony tests suggest the possible presence of resistance factors in both the serum and the mucus at later
stages of trematode infection, and that these factors
result in recovery from the infection.
The results also
suggest that resistance factors in the mucus may originate from the serum.
Although the resistance factors were not characterized, they probably have an immunological function.
Immunological factors that are found in both the serum
and mucus of fish include: immunoglobulin (Fletcher and
Grant, 1969; Harris 1972; Bradshaw et al., 1971); complement (Harrell et al., 1976); lysozyme (Fletcher and
Grant, 1968); and C-reactive protein (Ramos and Smith,
1978).
Thus far, only IgM-like, precipitating antibodies
(plus complement) have been indicated in resistance to
helminth infections (Evans and Gratzek, 1989; McVicar and
Fletcher; 1970; Cottrell, 1977; Harris 1972).
Quantifying and characterizing the resistance factor
that appears to be present in English sole serum and
mucus may give us information that would allow us to
better understand mechanisms involved in resistance to
monogenetic trematodes.
The factor could be quantified
by obtaining a titer of the precipitins in each mucus
sample, this would indicate if there 'were differences in
72
the amount of precipitins present at different stages of
infection.
Separating the different components of the mucus by
column chromatography and repeating the mucus bioassays
on the fractions would be the first step in isolating and
identifying the factor responsible for resistance.
Once
the fraction containing the resistance factor was isolated, the factor itself could be identified through biochemical analyses.
Biochemical analyses would also
indicate if the same factor was in the serum and the
mucus.
If the factor was determined to be an immunoglobulin, then the presence of complement activity
could be tested by repeating the bioassays with heattreated mucus and serum.
The Ouchterlonys should be
repeated with the resistant fraction of the mucus to
determine if the factors that precipitated in whole mucus
samples were contained in the resistant fraction.
73
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